The Myc oncoprotein is overexpressed in a wide variety of human tumors. In order to maintain normal levels of Myc protein in quiescent cells, newly synthesized Myc is rapidly degraded via the ubiquitin/proteasome pathway. Ubiquitin-mediated degradation of Myc is cell growth regulated, dependent upon the action of two Ras effector pathways. Specifically, Ras activated Raf-MEK-ERK-mediated phosphorylation of Myc at Serine 62 stabilizes Myc, whereas phosphorylation of Myc at Threonine 58 by GSK-3Beta, which is dependent on prior phosphorylation of Serine 62 and is regulated by Ras activated PI3K-Akt, stimulates Myc degradation. In addition, loss of Serine 62 phosphorylation precedes ubiquitination of Myc and the Protein Phosphatase 2A (PP2A) is responsible for this dephosphorylation. PP2A is a conformation-specific phosphatase whose activity is enhanced by the phospho-specific prolyl isomerase Pin1. Pin1 facilitates the removal of the Serine 62 phosphate in Myc by PP2A, thus promoting Myc degradation. These observations have defined a cascade of programmed events that insure the transient accumulation of Myc during the initial phase of a cellular growth response. This grant proposal is intended to further elucidate cellular mechanisms that regulate Myc protein stability, with the goal of better understanding Pin1 and PP2A actions on Myc and discovering other cellular proteins that specifically promote Myc degradation. These Myc-degrading proteins will then be used to inhibit Myc-induced cellular transformation. The following specific aims will be pursued: 1. Investigate cellular mechanisms regulating Myc protein stability, 2. Analyze Pin1 and PP2A regulation of Myc degradation and their contribution to Myc overexpression in human tumors, 3. Identify the E3 ubiquitin ligase involved in Myc degradation and study the effects of forced Myc degradation on Myc-mediated oncogenesis. Research has previously shown that inhibition of one of the cellular events in the genesis of cancer can revert tumor formation. Thus, analysis of molecular mechanisms that control Myc accumulation and identification of proteins that specifically target Myc for degradation could be instrumental in developing specific inhibitors of Myc, with the ultimate goal of designing a therapeutic approach for human cancers that are dependent upon high levels of Myc protein.